1. Field of the Invention
The present invention relates to a method for enriching antisense messenger RNA that are naturally expressed in cells.
2. Description of the Related Art
The functional information of the genes in the genome is unidirectional. Taking as an example the most simple gene which encodes a protein, the RNAs encoding a protein are transcribed from one of the strands and define one mRNA that possesses the information for the translation of a single protein. This simplistic view is obviously more complex for many genes due to mechanisms such as alternative splicing and intron encoded mRNAs. While this general view of the unidirectional nature of genes is true with regard to structural information, it is not so with regard to regulatory information. The possibility of transcription of the other strand of a gene, in the reverse direction, will result in the production of an RNA that is complementary to the mRNA that possesses the structural information (the sense mRNA). Such RNAs, termed antisense RNAs, have the potential to strongly bind to the sense mRNA, producing a double stranded RNA, and to inhibit the realization of the structural information. However, it is possible that these RNAs also possess structural information.
In general, natural antisense RNAs are endogenous transcripts containing regions complementary to transcripts of other genes or other transcripts arising from the same gene locus. Antisense RNA is an RNA which contains a stretch of nucleotides complementary to another RNA that has some cellular function. The length of the complementary stretch is usually a few hundred nucleotides, but shorter stretches can also be important. The expression of antisense RNA is a powerful way of regulating the biological function of the sense RNA molecules. Natural antisense RNAs have been shown to play important regulatory roles, including control of cell growth, malignant transformation and other cellular phenotypes. Through the formation of a stable duplex between the sense RNA and antisense RNA, the normal or sense RNA transcript can be rendered inactive and untranslatable.
Natural antisense transcripts can either be cis-encoded or trans-encoded. Cis-encoded antisense arises from transcription of the complementary strand of the sense gene; both sense and antisense transcripts originate from the same locus and thus, the antisense transcripts have regions of perfect complementarity to the sense mRNA. Trans-encoded antisense arises from transcription of a different genomic locus, and accordingly, it is expected that in such cases the complementarity of the antisense region will be not complete.
Following the discovery of natural antisense RNAs in prokaryotes, natural antisense RNAs were also discovered in a variety of eukaryotes covering a wide range of the phylogenetic tree, including viruses (Michael et al., 1994), slime molds (Lee et al., 1993), insects (Lankenau et al., 1994), amphibians (Kimelman et al., 1989), birds (Farrell et al., 1995) and mammals (Murphy et al., 1994). Moreover, most of the genes for which endogenous antisense transcripts were discovered encode proteins of key regulatory roles in important cellular phenotypes, such as cellular proliferation (Chang et al., 1991), apoptosis (Khochbin et al., 1989) and embryonic development (Bedford et al., 1995), or of key cellular processes such as translation (Noguchi et al., 1994), transcription (Krystal et al., 1990) and splicing (Fu et al., 1992). The thorough review article by Vanhee-Brossollet and Vaquero (1998) offer a summary of all antisense RNAs discovered so far and their functional importance. The evidence suggests that control of gene expression by endogenous antisense RNAs is one of the regulatory mechanisms in the cell and is widespread throughout the eukaryotic kingdom.
All of the antisense transcripts discovered so far were found by sporadic experiment stemming from studies of single genes. This implies that the antisense regulatory mechanism might be of general importance and relevant for many more genes. Thus, a general method for finding those mRNAs in the cell for which an antisense RNA exists would be of great value. The object of the approach and method of the present invention is to enable those in the art to investigate which mRNAs in the cell have antisense RNAs, as compared to studies done up until now which investigated the question xe2x80x9cdoes this specific mRNA have an antisense RNA counterpart?xe2x80x9d
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
The present invention is directed to a rapid method for enrichment of natural antisense mRNA (the exonuclease and polymerase activities of DNA polymerases on the sense-antisense double-stranded hybrid), followed by amplification and cloning of its corresponding cDNA. Thus, the invention overcomes the deficiencies in the prior art as discussed above. This method provides for the enrichment and detection of natural antisense mRNA from any natural source of RNA. Poly A+ mRNA in a sample of RNA is converted into single-stranded cDNA which is denatured to disrupt any secondary structure, and then allowed to re-anneal under stringent conditions with any other cDNA having a segment with a significant complementary sequence to form a hybrid molecule with a double-stranded cDNA segment. Sense and antisense cDNAs hybridize to each other and form double-stranded stretches or segments of DNA. In the present method, the resulting hybridization products with double-stranded DNA segments are treated with a DNA polymerase, such as T4 DNA polymerase, which has a 5xe2x80x2 to 3xe2x80x2 polymerase activity and a 3xe2x80x2 to 5xe2x80x2 exonuclease activity. The resulting double-stranded molecules are then amplified and cloned.